title: Polymyxin B Induces Apoptosis in Kidney Proximal Tubular Cells.
abstract: The nephrotoxicity of polymyxins is a major dose-limiting factor for treatment of infections caused by multidrug-resistant Gram-negative pathogens. The mechanism(s) of polymyxin-induced nephrotoxicity is not clear. This study aimed to investigate polymyxin B-induced apoptosis in kidney proximal tubular cells. Polymyxin B-induced apoptosis in NRK-52E cells was examined by caspase activation, DNA breakage, and translocation of membrane phosphatidylserine using Red-VAD-FMK [Val-Ala-Asp(O-Me) fluoromethyl ketone] staining, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and double staining with annexin V-propidium iodide (PI). The concentration dependence (50% effective concentration [EC50]) and time course for polymyxin B-induced apoptosis were measured in NRK-52E and HK-2 cells by fluorescence-activated cell sorting (FACS) with annexin V and PI. Polymyxin B-induced apoptosis in NRK-52E cells was confirmed by positive labeling from Red-VAD-FMK staining, TUNEL assay, and annexin V-PI double staining. The EC50(95% confidence interval [CI]) of polymyxin B for the NRK-52E cells was 1.05 (0.91 to 1.22) mM and was 0.35 (0.29 to 0.42) mM for HK-2 cells. At lower concentrations of polymyxin B, minimal apoptosis was observed, followed by a sharp rise in the apoptotic index at higher concentrations in both cell lines. After treatment of NRK-52E cells with 2.0 mM polymyxin B, the percentage of apoptotic cells (mean ± standard deviation [SD]) was 10.9% ± 4.69% at 6 h and reached plateau (>80%) at 24 h, whereas treatment with 0.5 mM polymyxin B for 24 h led to 93.6% ± 5.57% of HK-2 cells in apoptosis. Understanding the mechanism of polymyxin B-induced apoptosis will provide important information for discovering less nephrotoxic polymyxin-like lipopeptides.
authors.affiliation: Drug Delivery, Disposition and Dynamics.
authors.forename: Mohammad A K
authors.initials: MA
authors.lastname: Azad
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors.forename: Ben A
authors.initials: BA
authors.lastname: Finnin
authors.affiliation: Drug Delivery, Disposition and Dynamics.
authors.forename: Anima
authors.initials: A
authors.lastname: Poudyal
authors.affiliation: Drug Delivery, Disposition and Dynamics.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Davis
authors.affiliation: Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria, Australia.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Anatomical Pathology, St Vincent's Hospital, Fitzroy, Victoria, Australia.
authors.forename: Prue A
authors.initials: PA
authors.lastname: Hill
authors.affiliation: Drug Delivery, Disposition and Dynamics.
authors.forename: Roger L
authors.initials: RL
authors.lastname: Nation
authors.affiliation: Drug Delivery, Disposition and Dynamics Jian.Li@monash.edu Tony.Velkov@monash.edu.
authors.forename: Tony
authors.initials: T
authors.lastname: Velkov
authors.affiliation: Drug Delivery, Disposition and Dynamics Jian.Li@monash.edu Tony.Velkov@monash.edu.
authors.forename: Jian
authors.initials: J
authors.lastname: Li